Abstract

In the ongoing quest to further expand the repertoire of drugs for cancer therapy, sights have turned on the machinations of the anti-malarial drug, chloroquine as an untapped prospective drug for use in such treatments.
In order to establish the nature of chloroquine diphosphate (CQ) on differing breast cancers subtypes and to explore its relationship with tamoxifen (TAM) both in comparative effect and in conjunction, three phenotypically disparate cell lines were investigated; oestrogen receptor positive (ER+) MCF-7 WT, tamoxifen resistant subtype MCF-7 TMX and oestrogen receptor negative (ER-) MDA-MD-231. In order to establish a baseline, each cell line was investigated first independently with various concentrations of tamoxifen (2-30 μM) and chloroquine (10-140 μM) for 48 hours, the results of which were used to design a combined treatment modality to assay the effect of both drugs on the cells in tandem. The preliminary results showed that response to the initial two single drug treatment studies were dose dependent with lower concentrations of tamoxifen (2-10 μM) and chloroquine (10-20 μM) inhibiting growth and increasing vacuolation in all cell lines evidenced by increased uptake in Neutral Red (NR) stain and increased volume of acidic compartments (VAC). Higher treatment concentrations (12-32 μM TAM, 40-120 μM CQ) were seen to have a cytotoxic effect on all the cell lines in conjunction with a decrease in NR uptake and VAC. All three cell lines exhibited analogous response to CQ and singular treatment with tamoxifen resulted in similar dose-dependent responses albeit with higher doses of the drug necessary for MCF-7 TAM and MDA-MB-231 to develop any significant (P≤ 0.05, n=3) cytotoxic deviation. This effect on the ER- and oestrogen resistant cell lines imply exogenous cytotoxic pathways independent of the oestrogen receptor. Combination treatment, using the same range of TAM with a static dose (10 μM) Studies combining the two drugs were performed using the preliminary data as rationale for dosage. Using variable concentrations of tamoxifen (2-30 μM) and keeping that of CQ (10 μM) saw changes in cell morphology mimicking more closely that of high dose CQ treatment in qualitative NR and acridine orange (AO) staining from relatively low doses of TAM as well as substantially decreasing the ED50 (minimum dosage to kill 50% of cells) in all cell lines. Caspase 3 colorimetry and Annexin V ELISA was used for the quantification of apoptosis and to help determine the mechanism of cell death utilised by these drugs and implied Caspase dependent apoptosis was occurring alongside the more visible caspase-independent cytotoxic events.
The results suggest that CQ may potentiate cell death of breast cancer cells treated with tamoxifen through the abrogation of autophagy and potentially re-sensitising resistant and ER- cells to hormone therapy treatment.